Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions

Interleukins are secreted proteins that regulate immune responses. Among these, the interleukin 12 (IL-12) family holds a central position in inflammatory and infectious diseases. Each family member consists of an α and a β subunit that together form a composite cytokine. Within the IL-12 family, IL-35 remains particularly ill-characterized on a molecular level despite its key role in autoimmune diseases and cancer. Here we show that both IL-35 subunits, IL-12α and EBI3, mutually promote their secretion from cells but are not necessarily secreted as a heterodimer. Our data demonstrate that IL-12α and EBI3 are stable proteins in isolation that act as anti-inflammatory molecules. Both reduce secretion of proinflammatory cytokines and induce the development of regulatory T cells. Together, our study reveals IL-12α and EBI3, the subunits of IL-35, to be functionally active anti-inflammatory immune molecules on their own. This extends our understanding of the human cytokine repertoire as a basis for immunotherapeutic approaches.


Fig. S2.
Recombinant IL-12a C96S and EBI3 are pure and interaction-competent proteins.(A) Detailed purification strategy with the corresponding Coomassie stained SDS-gels of IL-12a C96S and (B) EBI3.Pooled fractions are shown in rectangles.For EBI3, the red dashed line is shown to guide the eye for the comparison of its migration behavior under reducing versus non-reducing conditions.Note that the gel showing purified IL-12a C96S (top right) is the same as in main Figure 3A.(C) Recombinant IL-12a C96S and EBI3 interact with their partner subunits to form IL-12 and IL-27, respectively.IL-12a C96S and IL-12b C199S,His or EBI3 and murine IL-27a His were incubated and co-immunoprecipitation using the His-tag which reveals specific interaction.Murine IL-27a was used for all experiments were needed since human IL-27a cannot be produced in isolation.

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Fig. S3.Recombinant IL-12a C96S and EBI3 do not detectably interact to form IL-35. (A) Analytical ultracentrifugation experiments of IL-12aC96S and EBI3 confirm a mostly monomeric state of both proteins, with frictional ratios of 1.31 (IL-12a C96S ) and 1.45 (EBI3) and calculated molecular weights for the monomers of 27.3 kDa (IL-12a C96S ) and 27.5 kDa (EBI3).Some homodimers (51.4 kDa) are detected for EBI3.Incubation of both proteins did not indicate the formation of a heterodimeric complex.(B) Individually purified IL-12a C96S,His and EBI3 do not coimmunoprecipitate in contrast to EBI3 and mIL-27a His .(C) Hydrogen/deuterium exchange (HDX) experiments reveal IL-12a C96S stabilization through complex formation only after incubation with IL-12b C199S in contrast to EBI3.IL-12a C96S is colored according to the fractional uptake of HDX measurements.Blue color indicates a low (less flexible and potentially shielded regions) and red colors a high (flexible and solvent accessible regions) fractional uptake (gray: no sequence coverage in HDX measurements).

Fig. S4 .
Fig. S4.Functional effects of IL-12 C96S , EBI3 and control cytokines.(A) Gene expression of IL1B, IL6, CXCL8, TNFA and TGFB (qPCR) from LPS-stimulated human PBMCs after additional treatment with IL-12 C96S or EBI3 (n = 6 to 10 donors).(B) Gene expression of IL6 and TNFA (qPCR) from LPS-stimulated human PBMCs after additional treatment with IL-35 or the combination of IL-12a C96S and EBI3 (n = 6 donors).(C) Concentrations of IL-1b, IL-6 and IL-8 (ELISA) in culture supernatants produced by human MDM (n = 10 donors) after stimulation with HDM alone or after additional treatment with IL-35 or the combination of IL-12a C96S and EBI3.(D) Concentrations of IL-1b, IL-6, IL-8 and TNFa (ELISA) in culture supernatants produced by human PBMCs (n = 3 donors) after stimulation with HDM alone or after additional treatment with IL-10 or IL-27.Dotted line indicates mean secretion or gene expression of PBS treated PBMCs or MDMs.Data are presented as individual values.Donor dependent effect is shown by connecting line.Statistical significance was determined by Wilcoxon test.*p< 0.05; **p < 0.01.

Fig. S5 .
Fig. S5.Effects of IL-12 C96S , EBI3 and control cytokines on Treg cells and type 2 immunity.(A) Percentage of CD25 hi Foxp3 + Treg cells (FACS) in human PBMC cultures treated with increasing concentrations of IL-12a C96S or EBI3 (10-100 ng/ml) (n = 7).Data is presented as means + SEM.Statistical significance was determined by Friedmann test.*p < 0.05; **p < 0.01.(B) Gene expression of IL5 (qPCR) from LPS stimulated human PBMCs after additional IL-12a C96S or EBI3 treatment (n = 7 donors).(C) Concentration of CCL17 (ELISA) in culture supernatants produced by human PBMCs (n = 3 donors) after stimulation with IL-4 alone or after additional treatment with IL-10, IL-27 and IL-35.(B-C) Dotted line indicates mean secretion or gene expression of PBS treated PBMCs.Data are presented as individual values.Donor dependent effect is shown by connecting line.Statistical significance was determined by Wilcoxon test.*p< 0.05.

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Fig. S6.Testing CD81 interactions in IL-35.CD81 does not co-immunoprecipitate with IL-12a and EBI3.Co-immunoprecipitation of FLAG-tagged IL-12a with HA-tagged EBI3 in cell lysates and in the medium verifies assembly for these two proteins.Endogenous CD81 can be detected in the input lysate fraction only.